Sensitive detection of bacteria by improved nested polymerase chain reaction targeting the 16S ribosomal RNA gene and identification of bacterial species by amplicon sequencing

Abstract

A method for identifying an RNA form of a bacteria, comprising reverse transcribing RNA material; conducting PCR using primers for a first highly conserved genetic sequence generic of the bacteria; conducting nested PCR using primers for a second highly conserved genetic sequence within the first genetic sequence of the bacteria; and identifying the bacteria based on unconserved amplified sequences linked to the conserved sequences.

What is claimed is: 
     
       1. A method for amplifying genetic sequences, comprising the steps of:
 (1) first, reverse transcribing RNA to DNA, and conducting PCR using at least one outer primer having a sequence:
 SEQ ID NO: 1 (AAYGGGTGAGTAACACGT) for Gram positive bacteria or, 
 SEQ ID NO: 8 (RAYGGGTGAGTAAYGYMT) for Gram negative bacteria, and at least one primer having a sequence: 
 SEQ ID NO: 5 (CCCGRGAACGTATTCACSG), 
 
 (2) second, conducting nested PCR, using inner primers, comprising at least one primer having a sequence:
 SEQ ID NO: 6 (CTACGGGAGGCWGCAGTRRGGAAT), and at least one primer having a sequence: 
 SEQ ID NO: 7 (WGGGTATCTAATCCTRTTTGMTCCCCW), wherein R=G or A, S=G or C, W=A or T, M=A or C, and Y=T or C. 
 
 
     
     
       2. The method according to  claim 1  further comprising the step of identifying an organism corresponding to an amplicon resulting from said nested PCR step. 
     
     
       3. A kit, comprising a unit amount of each of the following combinations of primer sequences, substantially absent interfering DNA primer sequences, sufficient for a PCR identification of a bacteria in a biological sample:
 (a) (i) at least one sequence selected from the group consisting of:
 SEQ ID NO: 1 (AAYGGGTGAGTAACACGT) and SEQ ID NO: 8 (RAYGGGTGAGTAAYGYMT), and 
 (ii) SEQ ID NO: 5 (CCCGRGAACGTATTCACSG); and 
 
 (b) SEQ ID NO: 6 (CTACGGGAGGCWGCAGTRRGGAAT), and SEQ ID NO: 7 (WGGGTATCTAATCCTRTTTGMTCCCCW), 
 wherein R=G or A, S=G or C, W=A or T, M=A or C, and Y=T or C, 
 wherein said component (a) further comprises a reverse transcriptase (RNA-dependent, DNA polymerase) activity, and deoxynucleotide triphosphates. 
 
     
     
       4. The kit according to  claim 3 , wherein said component (b) further comprises temperature resistant, DNA-dependent DNA polymerase activity, and deoxynucleotide triphosphates. 
     
     
       5. The kit according to  claim 3 , wherein at least one of said component (a) and (b) comprises 5 mM MgCl2, 50 mM Tris, pH 8.0, 15 mM (NH4)2SO4, 10 mM B-Mercaptoethanol, 500 μM dATP, dCTP, dGTP, and dTTP, 0.025% BSA, 1 μM of each primer, a reverse transcriptase (RNA dependent DNA polymerase) activity, and a temperature resistant, DNA-dependent DNA polymerase activity. 
     
     
       6. A method for using the kit according to  claim 3  for amplifying genetic sequences, comprising the steps of:
 (1) first, reverse transcribing RNA to DNA, and conducting PCR using at least one outer primer having a sequence:
 SEQ ID NO: 1 (AAYGGGTGAGTAACACGT) for Gram positive bacteria or, SEQ ID NO: 8 (RAYGGGTGAGTAAYGYMT) for Gram negative bacteria, and at least one primer having a sequence: 
 SEQ ID NO: 5 (CCCGRGAACGTATTCACSG), 
 
 (2) second, conducting nested PCR, using inner primers, comprising at least one primer having a sequence:
 SEQ ID NO: 6 (CTACGGGAGGCWGCAGTRRGGAAT), and at least one primer having a sequence: 
 SEQ ID NO: 7 (WGGGTATCTAATCCTRTTTGMTCCCCW), 
 
 wherein R=G or A, S=G or C, W=A or T, M=A or C, and Y=T or C. 
 
     
     
       7. The method according to  claim 6  further comprising the step of identifying an organism corresponding to an amplicon resulting from said nested PCR step.